Hi,
I'm currently working with an enzymatic reaction. The reagents are quite expensive, so I wonder if I can scale down the reaction volume from 100ul to 25ul. Is this possible or there is any consideration about these kind of issues?
Martin:
Are you trying to simply run the reaction on a smaller scale, but with the relative amounts of everything the same ( this should be fine) or are you trying to increase the concentration or change the reelative ratios ( not such a good idea)
Mitch
Mitchell, I'm not changing the relative ratios. Changing eppendorf to plate assay could alter the reaction?
If all your reagents and your enzyme are soluble, that should be fine- I have run reactions in 96-well plates that had previously been done in tubes without an issue- keep the plate covered to minimize evaporative losses, especially at elevated temperatures or extended reaction times.
A potential problem with switching to microplates is the loss of the enzyme to adsorption to the plate surface. This is often a problem with polystyrene plates, not so much with polypropylene plates. To prevent it, add some detergent to your buffer. The detergent blocks the polystyrene. I use 0.005 or 0.01% Triton X-100, but many nonionic detergents can be used, including Tween-20 and Brij-35. The detergent concentration does not have to be above the critical micellar concentration.
If you have the capability to use 384-well plates, the reaction volume can be reduced even further.
If you are measuring absorbance, keep in mind that the absorbance is directly proportional to the path length, which is directly proportional to the volume in the well (for flat-bottom wells). If you are measuring fluorescence or luminescence, this is not the case, allowing very small volumes (e.g. 6 µl) to be used in special low-volume 384-well plates.
I would like to recommend an HPLC-enzyme assay, which uses small amount of high-cost substrate (please see file; J Chrom B rat BIN LIP).
It depends on whatever assay you use and its resolution. For reaction, reduction enzyme by volume or concentration should be fine, but the signal, whatever assay you use, will scale down. For example, in HPLC, if the peak is too small, it is hard to get reliable integration. Therefore, you just need to experiment with it to find workable amount of enzyme.
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